Proteinase-free coatings for colony passaging

ABSTRACT

A cell culture article includes a substrate having a polymer coating that is conducive to colony passaging of cells cultured on the coating. Example polymer coatings are formed from polygalacturonic acid (PGA), alginate, or combinations thereof. Cells cultured on the polymer coating can be separated from the substrate as a colony or layer of cells by exposing the polymer coating to (i) a chelating agent, (ii) a proteinase-free enzyme, or (iii) a chelating agent and a proteinase-free enzyme.

This application claims the benefit of priority under 35 U.S.C. § 119 ofU.S. Provisional Application Ser. No. 62/095147 filed on Dec. 22, 2014the content of which is relied upon and incorporated herein by referencein its entirety.

BACKGROUND Field

The present disclosure relates generally to articles and methods forcell dissociation, and more specifically to polymer-coated substratesand related protocols for colony passaging of human stem cells.

Technical Background

Stem cell research is a rapidly advancing field with the potential todevelop therapeutic agents to treat diseases as well as study diseasedevelopment. The culture of human stem cells shares many of the sameprotocols as standard mammalian cell culture. However, the successfulculture and maintenance of human stem cells, including inducedpluripotent stem cells (IPSCs) and human embryonic stem cells (hESCs) inan undifferentiated state requires additional considerations to ensurethat cells maintain their key characteristics of self-renewal andpluripotency.

Successful stem cell culture benefits from the re-creation of an in vivostem cell microenvironment, which includes growth factors, cell-to-cellinteractions, and cell-to-matrix adhesions. Unlike many cell types,human stem cells are grown in aggregates, or colonies, which helpscreate this microenvironment.

Conventional culture of human stem cells involves exposure to mediaenriched with growth factors found in fetal bovine serum (FBS) ordefined serum replacements, for example. Further, such human stem cellculture systems may utilize support cells such as an inactivated mouseembryonic fibroblast (MEF) feeder layer to support growth and preventdifferentiation. These cells provide intercellular interactions,extracellular scaffolding, and factors creating a robust and stable cellculture environment.

There are several fundamental aspects involved in the culturing ofcells, including thawing frozen stocks, plating cells in culturevessels, changing media, passaging and cryopreservation. Passagingrefers to the removal of cells from one culture vessel and theirsubsequent transfer to one or more new culture vessels. Passaging isadvantageous in minimizing the harmful effects of overcrowding and forpromoting expansion of the culture.

Traditional proteinase-based methods for harvesting cells typicallyproduce only single cells and may also adversely affect viability andstem character of those cells produced. During dissociation, the cellsare removed from a growth surface by scraping such as with a cellscraper or lifter. However, this process is labor intensive and resultsin cells having an unacceptably high degree of variability (e.g., colonysize, viability, etc.). Moreover, the use of a scraper is not suitablefor high density cell culture formats such as multi-layer culturevessels, roller bottles, or microcarriers.

In view of the foregoing, improved methods and apparatus for colonypassaging of stem cells would be beneficial.

BRIEF SUMMARY

In accordance with embodiments of the present disclosure, a substratefor culturing cells comprises a polymer coating disposed on a surface ofthe substrate. The polymer coating is cross-linked or grafted to thesubstrate and comprises at least one of PGA and alginate. The polymercoating may be cross-linked with calcium ions.

A method for culturing cells comprises forming a polymer coating on asubstrate surface, wherein the polymer coating comprises at least one ofPGA and alginate, forming a cell adhesion layer on the polymer coating,culturing cells on the cell adhesion layer, and separating the cellsfrom the cell adhesion layer as a colony or layer of cells by exposingthe polymer coating to (i) a chelating agent, (ii) a proteinase-freeenzyme, or (iii) a chelating agent and a proteinase-free enzyme.

Additional features and advantages of the subject matter of the presentdisclosure will be set forth in the detailed description which follows,and in part will be readily apparent to those skilled in the art fromthat description or recognized by practicing the subject matter of thepresent disclosure as described herein, including the detaileddescription which follows, the claims, as well as the appended drawings.

It is to be understood that both the foregoing general description andthe following detailed description present embodiments of the subjectmatter of the present disclosure, and are intended to provide anoverview or framework for understanding the nature and character of thesubject matter of the present disclosure as it is claimed. Theaccompanying drawings are included to provide a further understanding ofthe subject matter of the present disclosure, and are incorporated intoand constitute a part of this specification. The drawings illustratevarious embodiments of the subject matter of the present disclosure andtogether with the description serve to explain the principles andoperations of the subject matter of the present disclosure.Additionally, the drawings and descriptions are meant to be merelyillustrative, and are not intended to limit the scope of the claims inany manner.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of specific embodiments of thepresent disclosure can be best understood when read in conjunction withthe following drawings, where like structure is indicated with likereference numerals and in which:

FIG. 1A is a schematic diagram of a polymer-coated substrate and itsmethod of use according to one embodiment;

FIG. 1B is a schematic diagram of a substrate in which a grafted polymercoating is cross-linked using a divalent ion according to oneembodiment;

FIG. 1C is a schematic diagram of the polymer-coated substrate of FIG.1B with a cell adhesion layer according to one embodiment;

FIG. 1D is a schematic diagram of the polymer-coated substrate of FIG.1C with cells being adhered to the cell adhesion layer according to oneembodiment;

FIG. 1E is a schematic diagram of the polymer-coated substrate of FIG.1C with cells adhered to the cell adhesion layer according to oneembodiment;

FIG. 1F is a schematic diagram of the polymer-coated substrate in a stepof harvesting cells from the substrate according to one embodiment;

FIG. 2A is a schematic diagram of a polymer-coated substrate and itsmethod of use according to a further embodiment;

FIG. 2B is a schematic diagram of a polymer-coated substrate having acell adhesion layer according to an embodiment;

FIG. 2C is a schematic diagram of a polymer-coated substrate with cellsbeing adhered to a cell adhesion layer according to an embodiment;

FIG. 2D is a schematic diagram of a polymer-coated substrate with cellsadhered to a cell adhesion layer according to an embodiment;

FIG. 2E is a schematic diagram of the polymer-coated substrate in a stepof harvesting cells from the substrate according to one embodiment;

FIG. 3A is a schematic showing hypothetical cases of grafted polymers ona flat substrate in a brush configuration according to an embodiment;

FIG. 3B is a schematic showing hypothetical cases of grafted polymers ona flat substrate in a mushroom configuration according to an embodiment;

FIG. 3C is a schematic showing hypothetical cases of grafted polymers ona flat substrate in a mushroom configuration according to an embodiment;

FIG. 4 shows example polymer-coated substrate assemblies;

FIG. 5A shows absorbance results of gold staining for VN grafted ontoPGA;

FIG. 5B shows BCA results for VN grafted onto PGA;

FIG. 6 are phase contract micrographs illustrating ES-D3 cell adhesionon various surfaces according to embodiments;

FIG. 7 are phase contrast micrographs illustrating ES-D3 cell adhesionon various substrates according to embodiments;

FIG. 8 are phase contrast micrographs illustrating hMSC adhesion onvarious substrates according to embodiments;

FIG. 9A shows MTT quantification of cell number relative to thereference mesencult substrate plate;

FIG. 9B shows the efficiency of cell release using pectinase/EDTAtreatment;

FIG. 10 are absorbance results of crystal violet staining on PGA-basedsurfaces before and after pectinase/EDTA treatment;

FIG. 11 shows phase contrast micrographs illustrating the effect oftreatment with pectinase, EDTA, or the combination of pectinase andEDTA;

FIG. 12 is a chart quantifying hMSC growth according to variousembodiments;

FIGS. 13A and 13B show absorbance data and BCA data for cross-linked PGAor cross-linked PGA-PLL coatings provided with layers of eitherSynthemax+ or Synthemax II;

FIG. 14 shows phase contrast micrographs illustrating ES-D3 celladhesion on control and Synthemax+ coated PGA substrates;

FIG. 15 shows phase contrast micrographs illustrating hMSC cell adhesionon control and Synthemax+ coated PGA substrates;

FIG. 16 shows BCA data for coatings prepared with and withoutcross-linking using a cast and cure process;

FIG. 17 shows phase contrast micrographs illustrating ES-D3 adhesion oncontrol and mixed PGA/Synthemax coated substrates;

FIG. 18 shows phase contrast micrographs illustrating hMSC adhesion oncontrol and mixed PGA/Synthemax coated substrates;

FIG. 19A is BCA results and FIG. 19B is gold staining results for PGA-VNcoatings on TCT or PLL substrates for various PGA-VN concentrations;

FIG. 20 shows phase contrast micrographs illustrating ES-D3 adhesion oncontrol surfaces and surfaces coated with PGA-VN copolymer;

FIG. 21 shows phase contrast micrographs illustrating hMSC adhesion andgrowth on control surfaces and surfaces coated with PGA-VN copolymer;

FIG. 22 is quantitative data of cell growth and cell release for hMSCcells on control surfaces and surfaces coated with PGA-VN copolymer;

FIG. 23 shows phase contrast micrographs illustrating hMSC adhesion andgrowth on control surfaces and surfaces coated with the PGA-VN copolymerusing a KB process;

FIG. 24A is quantitative data for hMSC growth at day 5 on Mesencultsubstrate (MC substrate);

FIG. 24B is cell release data from different surfaces using pectinaseEDTA;

FIG. 25 shows hMSC release data using pectinase/EDTA for varioussolution concentrations, culture media and surface types; and

FIG. 26 shows (left) MTT quantification of cell growth at day 5 for HEKand MRCS cells on candidate surfaces, and (right) cell releaseefficiency for different cell release solutions.

DETAILED DESCRIPTION

Reference will now be made in greater detail to various embodiments ofthe subject matter of the present disclosure, some embodiments of whichare illustrated in the accompanying drawings. The same referencenumerals will be used throughout the drawings to refer to the same orsimilar parts.

Disclosed is a cell culture article that comprises a polymer coatingconducive to colony passaging of cells cultured on the coating.Exemplary cells include embryonic stem cells and pluripotent stem cells,including human embryonic stem cells (hESCs) and induced pluripotentstem cells (IPSCs), as well as other cell types that are beneficiallypassaged as colonies or clusters. Colony passaging is a favored approachfor preserving cell-to-cell associations that are important forpromoting, inter alia, cell self-renewal and genetic stability. Cellsmay be cultured directly on the polymer coating or on an interveninglayer provided between the cells and the polymer coating.

During cell harvesting, the polymer coating may be renderedun-cross-linked or at least partially digested, e.g., by aproteinase-free enzyme, to release the cells without damaging thecell-to-cell interactions within the colony or cell layer.

Example polymer coatings comprise polygalacturonic acid (PGA), alginate,and combinations thereof. Polygalacturonic acid, if used, may becross-linked or partially cross-linked such as with calcium.

The polymer layer may be provided on a substrate. The substrate may beany suitable support or vessel such as microcarriers, Petri dishes,bottles, beakers, flasks, and multi-layer vessels such as CellSTACK®culture chambers or HYPERflask® cell culture vessels. Because thepolymer layer (as well as any cell adhesion layer) can be applied towide variety of substrate geometries, the disclosed apparatus enablescolony dependent cell culture that is readily scalable in connectionwith protocols where manual scraping would not be possible.

One or more cell adhesion layers may be disposed at least partially overthe polymer layer in order to provide a cell-facing adhesion layer. Thecell adhesion layer(s), if provided, may comprise an intervening layerformed over the polymer coating, or may be integrated within the polymercoating such as by forming a polymer coating/adhesion layer mixture orco-polymer. The adhesion layer may be grafted (or covalently-bonded) tothe polymer coating. Example cell adhesion layers comprise extracellularmatrix (ECM) proteins, such as laminin, collagen or fibronectin, orsynthetic molecules such as poly-D-lysine or a Synthemax® surface, whichpromote cell attachment and growth. The cell adhesion layer(s) promotecell attachment and growth.

The architecture of example polymer-coated substrates is illustrated inFIGS. 1 and 2 in connection with their associated methods of useaccording to various embodiments. In one embodiment, the polymer coatingis grafted onto a substrate. The polymer chains may be grafted to thesubstrate at one or more sites along the polymer chain.

Example geometries of grafted polymers on a flat substrate surface areshown in FIGS. 3A-3C, where the space between molecules is 2/3 of s,where s is the mean distance between molecules. The other 1/3 of thereferenced length is occupied by the polymer itself. FIG. 3A shows themolecules in a brush configuration, while FIGS. 3B and 3C show themolecules in a mushroom configuration. The graft density of thepolymer(s) adsorbed onto the substrate may be 1/3, as illustrated, orgreater depending on the application. The graft density may range from33% to 100%, e.g., 33, 35, 40, 50, 60, 70, 80, 90 or 100%, includingranges between any of the foregoing. A thickness of the grafted polymercoating may range from 10 nm to 10,000 nm, e.g., 10, 20, 50, 100, 200,500, 1000, 2000, 5000 or 10,000 nm, including ranges between any of theforegoing. A grafted polymer coating may be un-cross-linked, partiallycross-linked or fully cross-linked. The polymer coating may be formed bygrafting PGA or alginate polymer, for example, to the substrate surfacethrough charge interaction or covalent bonding.

In the absence of (or prior to) cross-linking, the grafted polymer formsa highly hydrated, non-fouling surface. A partially cross-linked orfully cross-linked grafted polymer will exhibit reduced mobility, whichwill enhance its accessibility to proteins and cells. In embodiments, agrafted polymer coating is at least partially cross-linked prior to cullculture. The degree of cross-linking may range from 1 to 100 mol %,e.g., 1, 2, 5, 10, 20, 50, 60, 70, 80, 90 or 100 mol %, including rangesbetween any of the foregoing. Prior to cell exposure, a cell adhesionlayer is optionally formed over the polymer coating. Cells attach to andgrow on the polymer coating via the cell adhesion layer. The thicknessof a cell adhesion layer 400, when used, may range from 10 nm to 1micron, e.g., 10, 20, 50, 100, 200, 500 or 1000 nm, including rangesbetween any of the foregoing. The cell adhesion layer may completely orpartially cover the polymer coating.

With reference to FIG. 1A, illustrated is a substrate 100 having agrafted polymer coating 200 formed on a major surface 101 thereof. Thegrafted polymer coating 200 (e.g., after forming the grafted polymercoating on the substrate) may be cross-linked using a divalent ion suchas calcium, for example, as shown in FIG. 1B. Calcium ions 600 are ableto cross-link polymers such as PGA and alginate moieties because theycan form two bonds (as opposed to monovalent ions such as sodium, whichcan form only a single bond). Suitable sources of calcium ions includecalcium chloride and/or calcium carbonate. Cross-linking of the polymercoatings can minimize their dissolution into the cell culture medium.

Because calcium is incorporated into the polymer coating after itsformation, the degree of cross-linking of the coating 300 can benon-uniform, with a higher degree of cross-linking near the free surfaceof the polymer, and a lesser degree of cross-linking through the coatingthickness approaching the substrate.

Cross-linking is often measured by swelling experiments. A cross-linkedsample is placed into a solvent at a specified temperature, and eitherthe change in mass or the change in volume is measured. The extent ofcross-linking is inversely proportional to the extent of swelling. Basedon the degree of swelling, the Flory Interaction Parameter (whichrelates the solvent interaction with the sample), and the density of thesolvent, a theoretical degree of crosslinking can be calculatedaccording to Flory's Network Theory. ASTM Standard D2765 can be used tocalculate the degree of cross-linking.

As illustrated in FIG. 1C, a cell adhesion layer 400 may be formed overthe grafted polymer that has been cross-linked 300. Cultured cells 500bound to the cell adhesion layer 400 are shown schematically in FIGS. 1Dand 1E. With particular reference to FIG. 1E, cells 500 form a clusteror colony 550 on the surface of the cell adhesion layer.

To harvest the cells, EDTA is added to the media. With reference to FIG.1F, EDTA molecules 700 scavenge calcium ions 600 and return the polymercoating to a highly hydrophobic, non-fouling state. The cultured cellswill be released into the media as colonies or cell sheets 550. Releaseand harvesting of the cultured cells is performed in the absence ofproteinase.

The grafted polymer coating 200 in FIG. 1F can be recycled (i.e.,re-introduced to the protocol at FIG. 1A). The grafted polymer coatingcan be re-cross-linked and a new cell adhesion layer can be formed overthe grafted polymer coating in advance of a further cycle of cellattachment, cell growth and cell harvesting.

In a further embodiment, a 0.5 micron to 1000 micron thick polymercoating is uniformly cross-linked on a substrate. The thickness of thepolymer coating may be 0.5, 1, 20, 5, 10, 20, 50, 100, 200, 500 or 1000microns, including ranges between any of the foregoing. The degree ofcross-linking may range from 1 to 100 mol %, e.g., 1, 2, 5, 10, 20, 50,60, 70, 80, 90 or 100 mol %, including ranges between any of theforegoing.

The polymer coating may be formed by mixing a water solution of PGA oralginate with CaCO₃ powder to form a suspension that is applied to asurface of the substrate. The suspension may optionally include asurfactant or solvent (in addition to water) to promote the formation ofa thin coating. The coating is exposed to acetic acid vapor, whichreacts with the CaCO₃ to release Ca²⁺ ions that, in turn, bind to thePGA or alginate polymer and cross-link the polymer. Evaporation of thewater, surfactant and/or solvent may occur before, during or aftergelation (cross-linking).

Prior to introducing the cells and growth media, a cell adhesion layeris optionally formed over the cross-linked polymer coating. Cells attachto and grow on the polymer coating via the cell adhesion layer. Inembodiments, cells are cultured in direct physical contact with the celladhesion layer.

To harvest the cells, EDTA is added to the growth media. The EDTAscavenges the calcium and compromises the cross-linking of the polymer.Optionally, pectinase or alginate lysase may be introduced to the mediato proactively cleave the polymer bonds and expedite dissolution ordigestion of the polymer coating. The combination of cross-linkingannihilation by EDTA and cleavage by enzyme make the process very fast,which minimizes the negative impact to the cell product. As a result,cells will be released into the media as colonies or cell sheets. In theabsence of proteinase, the integrity of cell-to-cell interactions in thereleased cells is preserved.

In FIG. 2A is illustrated a substrate 100 having a cross-linked polymercoating 300 formed on a major surface thereof. Because calcium isincorporated in situ into the polymer coating, the degree ofcross-linking of the coating 300 can be uniform throughout the coatingthickness. As illustrated in FIG. 2B, a cell adhesion layer 400 may beformed over cross-linked polymer coating 300. Cultured cells 500 boundto the cell adhesion layer 400 are shown in FIGS. 2C and 2D. Individualcells 500 form a cluster or colony 550, as depicted in FIG. 2D.

With reference to FIG. 2E, pectinase or alginate lysase 800 inconjunction with EDTA 700 may at least partially digest the polymercoating to release the cell cluster 550 in tact from the substrate 100.The cell adhesion layer 400 may disassociate in conjunction withdigestion of the polymer layer.

The polymer-coated substrates disclosed herein enable cell expansion inany suitable growth medium. Example media include chemically-definedmedia, serum-containing media, and serum-free media. The polymer-coatedsubstrates can be used to culture cells as cell sheets, for example, fortissue engineering or organ reconstruction. Once cell growth iscomplete, EDTA or another chelating agent optionally in combination withan enzyme such as pectinase or alginate lysase, is used to un-cross-linkor at least partially digest the polymer coating such that the culturedcells are separated from the underlying substrate. Cell-to-cellinteractions are sustained such that the cluster or colony ismaintained.

As illustrated in FIG. 4, the herein-disclosed cell harvesting protocolmay be carried out by forming the polymer coating on the surface of aflask or a microcarrier bead.

EXAMPLES A. Grafting PGA-VN

As one approach to obtain a PGA polymer functionalized with peptide, weinvestigated the possibility of coating poly lysine plates (PLL) withPGA. The PGA is then cross-linked by the action of CaCl₂ and VN peptideis grafted using EDC/NHS.

Data obtained on the peptide surface shows a correlation between peptidedensity and the quality of cell adhesion. Chemical characterizationswere performed to define the grafting conditions that allow the highestpeptide density on the PGA surface. The impact of EDC/NHS ratio andpeptide concentration were evaluated.

FIGS. 5A and 5B show gold staining and BCA quantification of VN peptidegrafted on PLL-PGA as a function of EDC/NHS ratio and VN peptideconcentration.

From the FIG. 5 data, we can conclude that the surface with the highestpeptide density is obtained with a VN concentration of 2.5 mM usingEDC:NHS of 100 mM:100 mM.

The surfaces were tested with ES-D3 cells in a xeno-free medium. Thequality of adhesion of this cell line is generally correlated to peptideconcentration and facilitates evaluation of peptide availability.

Phase contrast microscopy images illustrating ES-D3 cell adhesion after18 hours on control and VN-grafted PGA surfaces as a function of peptideconcentration and EDC/NHS ratio are shown in FIG. 6.

PGA plates prepared with different peptide concentrations (1 mM and 2.5mM) grafted using different ratios of EDC/NHS (100:100 and 200:50) weretested and cell adhesion was compared to Matrigel™ matrix coated platesor Synthemax® surfaces. Cell morphology on PLL-PGA plates grafted with2.5 mM of VN peptide using EDC/NHS 100:100 is comparable with themorphology on the Synthemax® surface. Cell adhesion is observed forother conditions, but was inferior to the Synthemax® surface. Noadhesion is observed in the absence of the peptide.

The foregoing plates were tested with hMSC in a xeno-free medium (XFmedium). Good cell adhesion and cell growth were observed on all thesurfaces grafted with peptide.

A further test was conducted with ES-D3 cells to evaluate the impact ofthe PLL substrate and to compare PLL-PGA-VN (1 mM VN, EDC/NHS 100:100)plates with PGA-VN (1 mM VN, EDC/NHS 100:100). The results presented inFIG. 7, which shows phase contrast micrographs illustrating ES-D3 celladhesion at 18 hours on control and VN grafted PGA surfaces coated onPLL or TCT, indicate that the removal of PLL has no negative impact onES-D3 cell adhesion and improves slightly the spreading of the cells.

The foregoing plates were then evaluated with hMSC. The phase contrastmicroscopy data in FIG. 8, which illustrates hMSC adhesion and growthafter 5 days on control and VN grafted PGA surfaces, show that thegrowth obtained after 5 days on PGA-VN plates is slightly better thanwhat is observed on Synthemax® surfaces or Mesencult (MC) substratecoated plates. On PLL-PGA-VN plates, the cell growth observed is belowthe references. With reference to FIG. 9A, cell growth was quantifiedusing MTT assay. The data confirm the results of observations with cellgrowth on PGA-VN exceeding what is obtained on reference plates(Synthemax and Mesencult substrate). Cell release was attempted with apectinase/EDTA mix on all surfaces, and the efficiency of the cellrelease is summarized in FIG. 9B. The release is complete for the PGAbased surfaces, but does not reach more than 40% on the other surfacetypes.

PGA-based surfaces, in embodiments are designed to allow protease-freecell release with a pectinase/EDTA solution. The action of EDTA inducesa disruption of PGA cross-linking by chelation of calcium ions, and aperturbation of cell-cell and cell-ECM interactions, while pectinasedigests the PGA chains. The action of both compounds is associative; itdegrades the PGA-VN polymer and induces cell release.

The results presented in FIG. 10 demonstrate, using CV staining, thatthe PGA surface is degraded after pectinase/EDTA treatment.

FIG. 11 is phase contrast microscopy images illustrating the effect of a5 minute treatment with pectinase, EDTA, or pectinase EDTA mixtures onVN grafted PGA or on Mesencult™ substrate coated plates. A 5 minutetreatment with pectinase alone alters cell adhesion but is notsufficient to completely release the cells. EDTA treatment alone has aminor effect on cell adhesion. However, a 5 minute treatment with apectinase/EDTA mixture induces a complete release of the cell from thePGA based polymer, while the same treatment has no effect on cellsadhering to TCT on a Mesencult™ substrate.

Using the grafting approach to attach the cell adhesion layer to thepolymer coating, we developed a surface for which a PGA polymer iscoated on TCT. After cross-linking with CaCl₂, VN peptide is graftedusing EDC/NHS chemistry. As illustrated in FIG. 12, the performance ofthis surface for the growth of hMSC exceeds what is obtained withSynthemax® substrates or MesencultTM substrates (a bio-coating)irrespective of the culture medium: FBS containing medium, Mesencult™ XFmedium, or StemGro® (MesenGro®) medium.

In various embodiments, complete cell release is obtained in 5 minutesin the absence of proteases using a pectinase/EDTA solution.

B. Polymer Coatings and Adhesion-Promoting Layers

According to one embodiment, an adhesive layer comprisingVNARGPEGMAAcoHEMA copolymer (Synthemax+, positively charged) was formedon a cross-linked PGA polymer coating on PLL or TCT substrates. Chemicalcharacterization of the PGA surfaces coated with Synthemax+ wasperformed in comparison with Synthemax II (VNPEGMAAcoHEMA) coatings.Absorbance results of gold staining and BCA data are summarized in FIG.13.

The highest peptide density is obtained with Synthemax II, thoughcomparable cell growth is obtained for each of PGA and PLL-PGA.

The plates prepared were tested with ES-D3 cells. The results obtainedare presented in the phase contrast images of FIG. 14, which showsgenerally poor cell adhesion.

The same experiment was repeated with hMSC. As with the ES-D3 cells,poor cell adhesion was observed, as depicted in the phase contrastmicrographs of FIG. 15, which show hMSC adhesion and growth at day 5 oncontrol and Synthemax+ coated PGA surfaces formed on PLL or TCTsubstrates. Similar results were obtained when using Synthemax II inlieu of Synthemax+.

Synthemax layers on PGA polymer coatings provide surfaces with a highpeptide density (more than 10 pmol/mm² versus 5 pmol/mm² for thereference, Synthemax® R) but do not facilitate good cell adhesion.

Experiments to improve cell adhesion focused on a PGA/SynthemaxII blend.

According to a further embodiment, a PGA/SynthemaxII blend is used tocoat plates by adsorption (KB process or cast and cure), following whichthe polymer coating is cross-linked using CaCl₂.

For a cast and cure process, in order to see the impact of the Ca²⁺cross-linking a surface was prepared without a CaCl₂ incubation step.

FIG. 16 shows BCA results for surfaces prepared with and withoutcross-linking using the cast and cure process. The data indicate thatthere is no significant impact on the peptide concentration adsorbed onsurface.

The results obtained with ES-D3 cells are presented in FIG. 17. When thesurface is prepared with the KB process, cell adhesion is observed butis likely correlated to the adsorption of Synthemax polymer only. Withthe cast and cure process, however, good cell adhesion is observed whenthe polymer blend is not cross-linked with CaCl₂. Cell adhesion is lostafter cross-linking the polymer blend.

The foregoing surfaces were tested with hMSC, and the results of cellgrowth after 5 days are presented in FIG. 18. With the hMSC cells, aswith the ES-D3 cells, some adhesion and growth are observed with the KBprocess, and adhesion and growth is observed with the cast and cureprocess in the absence of cross-linking, but adhesion is lost when thepolymer blend is cross-linked.

Cell release was promoted with pectinase/EDTA. For the cast and cureplates, cells adhering and growing on the surface in the absence ofcross-linking cannot be released, while the few cells adhering oncross-linked surfaces are released efficiently by pectinase/EDTA. Thisobservation supports the hypothesis that the adhesion and growthobtained with this coating strategy is mainly an effect of the SynthemaxII polymer, and that if any PGA is involved it is not in a configurationthat promotes protease-free cell release. In FIG. 18, cell release fromPGA/Synthemax substrates using pectinase/EDTA is presented in the lowerrow.

In a further embodiment, a PGA-VN copolymer coating was prepared. TCT orPLL substrates were coated with different concentrations of PGA-VNpolymer using the cast and cure method and the polymer was cross-linkedwith CaCl₂ in water. BCA and gold staining results obtained with thisapproach are shown in FIGS. 19A and 19B respectively.

While there is no apparent impact on the gold staining results, BCAresults are slightly better with the PLL substrate and with the higherconcentration of 3 mg/ml.

Results obtained with ES-D3 cells in xeno free medium are presented inFIG. 20, which shows phase contrast micrographs illustrating ES-D3adhesion at 24 h on controls and surfaces coated with the PGA-VNcopolymer. The ES-D3 cells interact with PLL but adhesion and spreadingis poor on this surface. Adhesion is better in the presence of thePGA-VN polymer, and the results obtained are slightly better at 3 mg/mLand with a PLL pre-coating.

These plates were then tested with hMSC in mesencult XF xeno-freemedium. The phase contrast micrographs in FIG. 21 illustrate adhesionand growth at day 5 on controls and on the PGA-VN copolymer coatedplates. Cell repartition is more homogeneous on PLL precoated platesthan on TCT plates. Growth on the PLL PGA-VN plates is comparable towhat is obtained on Synthemax. Quantification of cell growth and cellrelease using pectinase EDTA are presented in the histograms of FIG. 22.

Cell quantification using MTT assay indicate that cell growth on PGA-VNis in the 80% range of what is obtained on Synthemax. Complete releaseis obtained on PGA-VN surfaces after treatment with pectinase/EDTA.

The cast and cure approach provides a decent performance level with hMSCin mesencult-XF, but obtaining a constant coating homogeneity isproblematic. In an attempt to improve homogeneity, a coating methodusing adsorption (KB process) was explored.

The KB process was evaluated on PLL pre-coated plates and on BDPureCoatAmine plates (PCA) with or without CaCl₂ crosslinking.

hMSC were grown for 5 days on plates in a mesencult XF medium. Phasecontrast micrographs illustrating hMSC adhesion and growth after 5 dayson control surfaces and surfaces coated with the PGA-VN copolymer usingthe KB process after CaCl₂ crosslinking or without CaCl₂ treatment areshown in FIG. 23. From the data in FIG. 23, the performance level iscomparable to Synthemax or mesencult substrates. The coating homogeneitywas slightly better on PCA plates than on PLL plates.

MTT cell growth data are summarized in FIG. 24 and confirm that cellgrowth on PCA PGA-VN is comparable to what is obtained on Synthemaxplates. Complete cell release is obtained with pectinase EDTA, butunexpectedly complete cell release is also obtained from Synthemax.

C. Effect of Enzyme/Chelating Agent Concentrations and Treatment Time onCell Release

The impact of pectinase/EDTA concentrations in protease-free cellrelease protocols from PGA surfaces and non-PGA surfaces wasinvestigated. Also evaluated was release from peptide grafted PGA withpectinase/EDTA in different media, including Mesencult XF, StemGro(MesenGro) and FBS containing media.

Data are summarized in FIG. 25. Referring first to the left-hand portionof the graph, a 5 minute treatment with 300 U pectinase/5 mM EDTAinduces release of a large proportion of the cells (e.g., at least 70%)under most conditions. The effect of EDTA alone is not sufficient toexplain this observation, since no cell release was observed with 5 mM.

The right-hand portion of the FIG. 25 graph, summarizes cell treatmentdata with 100 U pectinase /5 mM EDTA for KB-PGA-VN, grafted PGA-VN, andSynthemax II (Pas-1 usual KB) surfaces in different media. Interestinglywhile the release from KB-PGA-VN was only efficient in Mesencult XFmedium as previously described, release from grafted PGA-VN wascomplete, independent of the medium and more rapid than from KB-PGA-VN.With Synthemax II, only partial cell release was observed in theconditions evaluated. These data suggest that depending on conditions,pectinase may induce cell release via two mechanisms: a specificdegradation of the PGA polymer as observed with grafted PGA-VN surfaces,and an aspecific mechanism depending on culture conditions as observedon Synthemax II or KB-PGA-VN.

D. Cell Release using Pectinase/EDTA with HEK and MRCS Cell Lines

Pectinase/EDTA-induced cell release was evaluated for various surfaceswith HEK293 and MRCS cell lines. Cells were grown for 5 days onSynthemax II, Cellbind, KB-PGA-VN and grafted PGA-VN. Cell growth onthese different surfaces was quantified using MTT assays and the resultsare presented in FIG. 26 (left graphs). As seen with reference to thedata in FIG. 26 (right graphs) cell release was then explored using EDTA5 mM, pectinase alone, pectinase EDTA (100 U/5 mM), pectinase EDTA (300U/5 mM) or trypsin 0.25%.

As expected, for all cell lines trypsin induces complete releaseindependent of the type of surface. HEK cells release was unexpectedlyobtained from Synthemax and Cellbind with all pectinase containingsolutions. Release from grafted PGA-VN was expected but the resultsobtained with other surfaces do not suggest a specific singularmechanism. Interestingly, with KB-PGA-VN no complete release is obtainedfrom KB-PGA-VN plates. This suggests that the release obtained from thissurface with hMSC is a least partially aspecific.

With MRC5 cells, as expected, complete release is also obtained from allsurfaces with trypsin treatment, but grafted PGA-VN is the only surfacefrom which pectinase-containing solutions are able to induce a completerelease. These results indicate that the release obtained from graftedPGA-VN plates involves the degradation of the PGA polymer, but alsoindicate that pectinase is able to induce cell release with a variableefficiency depending on cell type and culture conditions through amechanism independent of PGA degradation.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a “polymer layer” includes examples having two ormore such “polymer layers” unless the context clearly indicatesotherwise

The term “include” or “includes” means encompassing but not limited to,that is, inclusive and not exclusive.

“Optional” or “optionally” means that the subsequently described event,circumstance, or component, can or cannot occur, and that thedescription includes instances where the event, circumstance, orcomponent, occurs and instances where it does not.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, examples include from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatany particular order be inferred. Any recited single or multiple featureor aspect in any one claim can be combined or permuted with any otherrecited feature or aspect in any other claim or claims.

It is also noted that recitations herein refer to a component being“configured” or “adapted to” function in a particular way. In thisrespect, such a component is “configured” or “adapted to” embody aparticular property, or function in a particular manner, where suchrecitations are structural recitations as opposed to recitations ofintended use. More specifically, the references herein to the manner inwhich a component is “configured” or “adapted to” denotes an existingphysical condition of the component and, as such, is to be taken as adefinite recitation of the structural characteristics of the component.

While various features, elements or steps of particular embodiments maybe disclosed using the transitional phrase “comprising,” it is to beunderstood that alternative embodiments, including those that may bedescribed using the transitional phrases “consisting” or “consistingessentially of,” are implied. Thus, for example, implied alternativeembodiments to a polymer layer comprising PGA and a cross-linking agentinclude embodiments where a polymer layer consists of PGA and across-linking agent and embodiments where a polymer layer consistsessentially of PGA and a cross-linking agent.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the present inventivetechnology without departing from the spirit and scope of thedisclosure. Since modifications, combinations, sub-combinations andvariations of the disclosed embodiments incorporating the spirit andsubstance of the inventive technology may occur to persons skilled inthe art, the inventive technology should be construed to includeeverything within the scope of the appended claims and theirequivalents.

1-8. (canceled)
 9. A method for making an article for culturing cells,comprising: forming a polymer coating on a substrate surface, whereinthe polymer coating is cross-linked with calcium ions and comprises atleast one of polygalacturonic acid (PGA) and alginate; and forming acell adhesion layer on the polymer coating, the cell adhesion layercomprising at least one of extracellular matrix (ECM) proteins andsynthetic molecules.
 10. The method according to claim 9, wherein thedegree of cross-linking is uniform across the polymer coating thickness.11. (canceled)
 12. The method according to claim 9, comprisingcross-linking the polymer coating after forming the polymer coating onthe substrate.
 13. (canceled)
 14. The method according to claim 12,wherein the degree of cross-linking decreases across the polymer coatingthickness in the direction of the substrate.
 15. A method for culturingcells, comprising: forming a polymer coating on a substrate surface,wherein the polymer comprises at least one of PGA and alginate; forminga cell adhesion layer on the polymer coating; culturing cells on thecell adhesion layer; and separating the cells from the cell adhesionlayer as a colony or layer of cells by exposing the polymer coating to(i) a chelating agent, (ii) a proteinase-free enzyme, or (iii) achelating agent and a proteinase-free enzyme.
 16. The method accordingto claim 15, wherein the chelating agent is EDTA.
 17. The methodaccording to claim 15, wherein the proteinase-free enzyme is pectinaseor alginate lysase.
 18. The method of claim 15, wherein the polymercoating is cross-linked with calcium ions.
 19. The method of claim 15,wherein the cell adhesion layer comprises at least one of extracellularmatrix (ECM) proteins and synthetic molecules.
 20. The method of claim9, wherein the polymer coating thickness ranges from 10 nm to 1000microns.
 21. The method of claim 9, wherein the substrate is selectedfrom the group consisting of microcarriers, dishes, bottles, beakers andflasks.